Figure 1. Principle of steric trapping and steric-trapping probes developed in this study.

(a) Steric trapping principle for measuring thermodynamic stability (ΔG^o_U) of proteins. After conjugation of biotin tags to two specific residues that are spatially close in the folded state but distant in the amino acid sequence, the first mSA binds unhindered to either biotin label with intrinsic binding affinity (ΔG^o_Bind). Due to the steric hindrance with pre-bound mSA, the second mSA binds only when the native tertiary contacts between biotinylated sites are unraveled by transient unfolding. Coupling of mSA binding to unfolding leads to attenuation of the apparent binding affinity of the second mSA relative to that of the first mSA, whose degree is correlated with the protein stability. Thus, thermodynamic stability of the target protein can be determined by fitting of the second binding phase (see equations (2)–(4) in Online Methods). Overall, protein unfolding is driven by the affinity and concentration of mSA without perturbing the native solvent condition. Folding reversibility is tested upon addition of excess free biotin by which bound mSA molecules are released by competition. (b) Thiol-reactive biotin derivatives possessing a spectroscopic reporter group developed in this study. BtnPyr-IA (1): biotin (red shaded)-pyrene (green shaded)-iodoacetamide (blue shaded) conjugated to a lysine template, and BtnRG-TP (2): biotin (red shaded)-1-oxyl-2,2,5,5-tetramethylpyrroline spin label (green shaded)-thiopyridine (blue shaded) conjugated to a cysteine template.